DNA quality requirements for Single-Molecule sequencing
Best practice DNA prep for SMRT Olga Vinnere Pettersson, PhD Project coordinator NGI-Sweden / SciLifeLab (UU)
National Genomics Infrastructure Sweden NGI Stockholm
NGI Uppsala
NGI staff: 60-70 FTE, including head of facility, lab research engineers, bioinformaticians, IT-experts, project coordinators. Operates since 1998 (separate nodes); from 2006 as an infrastructure. Hosted by
NGI-SciLifeLab is one of the most well-equipped NGS sites in Europe
10 17 3 1 2 6 2 2 1 2
Illumina HiSeq Xten Illumina HiSeq 2000/2500 Illumina MiSeq Illumina NextSeq Life Technologies Ion Torrent Life Technologies Ion Proton Pacific Biosciences RSII Sanger ABI3730 Argus Whole Genome Map. Syst. Oxford Nanopore MinIon
Our 2 machines have seen most of it… Applications: WGS De novo Iso-seq Short amplicons Long amplicons Metagenomics Sequence capture
Organisms: BACs, YACs, fosmids, plasmids, synthetics Gram positive bacteria Gram negative bacteria Archaea Parasitic protists Fungi (yeasts, moulds and mushrooms) Algae Mosses Higher plants Worms Butterflies Insects Birds Lizards Fish Mammals Human
WGS and De novo are the main applications => everything must go perfect We do not make the extractions; our users do
Sample looked good, but… WGS project of a yeast
And at the same time, another yeast WGS
260/280
260/230
1,9
2,07
1,91
2,15
1,9
2,14
WHY?! There is something in the DNA prep
• The entire workflow of PacBio De novo projects have been re-vised • Collection of successful protocols, tips & tricks started • Wrote a document • R&D on DNA quality assessment
Before the order is placed: • Meeting with the user • Sending a document with sample quality requirements • Going through the DNA extraction protocol, suggesting alterations and pointing out the most important steps. • Saving time and money in the sequencing step.
What makes PacBio so sensitive? Library prep is PCR-free It is a Single-molecule sequencing DNA-binders: • • • • •
Proteins Polyphenols Secondary metabolites (e.g. toxins) Pigments Polysaccharides
Polymerase inhibitors: • Salts • Phenol • Alcohols Physical inhibiting factors – debris
Hamilton & Arya, Nat. Prod. Rep., 2012, 29, 134-143
The DNA extraction process 1
2
What are the main contaminants? Polysaccharides Lypopolysaccharides Growth media residuals
Chitin Protein Secondary metabolites Pigments Growth media residuals
Chitin Fats Proteins Pigments
Polyphenols Polysaccharides Secondary metabolites Pigments
General DNA extraction Purpose
Chemistry
Disruption of bi-lipid membrane
Surfactants: SDS, CTAB, SLS, etc.
Inactivation of nucleases, removal of protein
Phenol : Chloroform : Isoamylalcohol 25:24:1 (0.6 vol)
Removal of phenol
Chloroform : Isoamylalcohol
Preferential precipitation of DNA
Ethanol (absolute) Isopropanol (Na-acetate)
Salt removal
Ethanol (70%)
Drying DNA precipitate
Speed-vac RT, +4°C
Re-suspending DNA precipitate
TE (1x), TE (1:10), water
Go back to pre-PCR molecular biology – search for protocols from 70s-90s
Recommended kit: Genomic Tip 10-500G QiaGen Gravity-flow, anion-exchange Average recovered molecule size: 50-100 kb
Other kits: MagAttract HMW DNA kit, QiaGen
Disclamer: Suggestions and advice are purely based on empirical evidence collected at Uppsala and that there is no conflict of interest.
Post-extraction clean-up: Use for samples contaminated with humic acids, phenol, lipids, secondary metabolites, etc. Recovers high-molecular weight DNA.
PowerClean®DNA clean-up kit MoBio Be prepared to lose up to 80% of the sample…
For every new DNA sample we ask for: • • • •
Gel picture 260/280 ratio 260/230 ratio Concentration estimated by TapeStation, Qubit, PicoGreen (be careful with NanoDrop), double of what is needed in the protocol.
• All users sign a consent form. • Samples that do not conform are rejected.
What does gel tell us? Protein contamination - Apply phenol-chloroform
Phenol carry-over or overloaded sample?
RNA contamination - Apply RNase, followed by phenol-chloroform extraction
If unsure, make dilution series. If problem persists – try MoBio clean-up kit, or re-extract DNA
What do absorption ratios tell us? Pure DNA 260/280: 1.8 – 2.0 < 1.8: Too little DNA compared to other components of the solution; presence of organic contaminants: proteins and phenol; glycogen - absorb at 280 nm. > 2.0: High share of RNA.
Pure DNA 260/230: 2.0 – 2.2 2.2: High share of RNA, very high share of phenol, high turbidity, dirty instrument, wrong blank. Photometrically active contaminants: phenol, polyphenols, EDTA, thiocyanate, protein, RNA, nucleotides (fragments below 5 bp)
Troubleshooting, Yeast Before:
BEFORE Alternations
260/280 = 1.9 260/230 = 2.2
Troubleshooting, Yeast After:
AFTER alternations
Troubleshooting: Mould BEFORE
AFTER
Before: Visible insoluble material in the sample, pigments. After: DNA collected with a glass rod directly after precipitation.
For every new de novo project with 8+ SMRT cells • We make a titration series on 4 SMRT cells to determine optimal loading of the library – Shearing test – Making a library (as long as possible) – Library QC and evaluating sizing information – Load 1x, 2x, 3x and 4x per SMRT cell – Determine the optimal concentration and % of reads above 10kbp – Adjusting calculations and quotes
DropSense R&D
Take home: • PCR-quality DNA ≠ PacBio quality DNA • Time spent on DNA extraction pays off • Go back to “ancient” protocols
Wish List: • New database over successful DNA extraction protocols • Continued R&D • Inter-lab collaboration
Special THANKS NGI / SciLifeLab, Uppsala
• • • •
Ida Höijer Susana Häggqvist Christian Tellgren-Roth Inger Jonasson
PacBio
• Jenny Ekholm • Swati Ranade
• Jamshid Fatehi
National Genomics Infrastructure Sweden NGI Stockholm
NGI Uppsala
NGI staff: 60-70 FTE, including head of facility, lab research engineers, bioinformaticians, IT-experts, project coordinators. Operates since 1998 (separate nodes); from 2006 as an infrastructure. Hosted by
NGI-SciLifeLab is one of the most well-equipped NGS sites in Europe
10 17 3 1 2 6 2 2 1 2
Illumina HiSeq Xten Illumina HiSeq 2000/2500 Illumina MiSeq Illumina NextSeq Life Technologies Ion Torrent Life Technologies Ion Proton Pacific Biosciences RSII Sanger ABI3730 Argus Whole Genome Map. Syst. Oxford Nanopore MinIon
Our 2 machines have seen most of it… Applications: WGS De novo Iso-seq Short amplicons Long amplicons Metagenomics Sequence capture
Organisms: BACs, YACs, fosmids, plasmids, synthetics Gram positive bacteria Gram negative bacteria Archaea Parasitic protists Fungi (yeasts, moulds and mushrooms) Algae Mosses Higher plants Worms Butterflies Insects Birds Lizards Fish Mammals Human
WGS and De novo are the main applications => everything must go perfect We do not make the extractions; our users do
Sample looked good, but… WGS project of a yeast
And at the same time, another yeast WGS
260/280
260/230
1,9
2,07
1,91
2,15
1,9
2,14
WHY?! There is something in the DNA prep
• The entire workflow of PacBio De novo projects have been re-vised • Collection of successful protocols, tips & tricks started • Wrote a document • R&D on DNA quality assessment
Before the order is placed: • Meeting with the user • Sending a document with sample quality requirements • Going through the DNA extraction protocol, suggesting alterations and pointing out the most important steps. • Saving time and money in the sequencing step.
What makes PacBio so sensitive? Library prep is PCR-free It is a Single-molecule sequencing DNA-binders: • • • • •
Proteins Polyphenols Secondary metabolites (e.g. toxins) Pigments Polysaccharides
Polymerase inhibitors: • Salts • Phenol • Alcohols Physical inhibiting factors – debris
Hamilton & Arya, Nat. Prod. Rep., 2012, 29, 134-143
The DNA extraction process 1
2
What are the main contaminants? Polysaccharides Lypopolysaccharides Growth media residuals
Chitin Protein Secondary metabolites Pigments Growth media residuals
Chitin Fats Proteins Pigments
Polyphenols Polysaccharides Secondary metabolites Pigments
General DNA extraction Purpose
Chemistry
Disruption of bi-lipid membrane
Surfactants: SDS, CTAB, SLS, etc.
Inactivation of nucleases, removal of protein
Phenol : Chloroform : Isoamylalcohol 25:24:1 (0.6 vol)
Removal of phenol
Chloroform : Isoamylalcohol
Preferential precipitation of DNA
Ethanol (absolute) Isopropanol (Na-acetate)
Salt removal
Ethanol (70%)
Drying DNA precipitate
Speed-vac RT, +4°C
Re-suspending DNA precipitate
TE (1x), TE (1:10), water
Go back to pre-PCR molecular biology – search for protocols from 70s-90s
Recommended kit: Genomic Tip 10-500G QiaGen Gravity-flow, anion-exchange Average recovered molecule size: 50-100 kb
Other kits: MagAttract HMW DNA kit, QiaGen
Disclamer: Suggestions and advice are purely based on empirical evidence collected at Uppsala and that there is no conflict of interest.
Post-extraction clean-up: Use for samples contaminated with humic acids, phenol, lipids, secondary metabolites, etc. Recovers high-molecular weight DNA.
PowerClean®DNA clean-up kit MoBio Be prepared to lose up to 80% of the sample…
For every new DNA sample we ask for: • • • •
Gel picture 260/280 ratio 260/230 ratio Concentration estimated by TapeStation, Qubit, PicoGreen (be careful with NanoDrop), double of what is needed in the protocol.
• All users sign a consent form. • Samples that do not conform are rejected.
What does gel tell us? Protein contamination - Apply phenol-chloroform
Phenol carry-over or overloaded sample?
RNA contamination - Apply RNase, followed by phenol-chloroform extraction
If unsure, make dilution series. If problem persists – try MoBio clean-up kit, or re-extract DNA
What do absorption ratios tell us? Pure DNA 260/280: 1.8 – 2.0 < 1.8: Too little DNA compared to other components of the solution; presence of organic contaminants: proteins and phenol; glycogen - absorb at 280 nm. > 2.0: High share of RNA.
Pure DNA 260/230: 2.0 – 2.2 2.2: High share of RNA, very high share of phenol, high turbidity, dirty instrument, wrong blank. Photometrically active contaminants: phenol, polyphenols, EDTA, thiocyanate, protein, RNA, nucleotides (fragments below 5 bp)
Troubleshooting, Yeast Before:
BEFORE Alternations
260/280 = 1.9 260/230 = 2.2
Troubleshooting, Yeast After:
AFTER alternations
Troubleshooting: Mould BEFORE
AFTER
Before: Visible insoluble material in the sample, pigments. After: DNA collected with a glass rod directly after precipitation.
For every new de novo project with 8+ SMRT cells • We make a titration series on 4 SMRT cells to determine optimal loading of the library – Shearing test – Making a library (as long as possible) – Library QC and evaluating sizing information – Load 1x, 2x, 3x and 4x per SMRT cell – Determine the optimal concentration and % of reads above 10kbp – Adjusting calculations and quotes
DropSense R&D
Take home: • PCR-quality DNA ≠ PacBio quality DNA • Time spent on DNA extraction pays off • Go back to “ancient” protocols
Wish List: • New database over successful DNA extraction protocols • Continued R&D • Inter-lab collaboration
Special THANKS NGI / SciLifeLab, Uppsala
• • • •
Ida Höijer Susana Häggqvist Christian Tellgren-Roth Inger Jonasson
PacBio
• Jenny Ekholm • Swati Ranade
• Jamshid Fatehi
